Pharmaceutical liquid composition of botulinum toxin with improved stability

ABSTRACT

Disclosed herein is a liquid pharmaceutical composition of  botulinum  toxin which is improved in stability. It comprises  botulinum  toxin, polysorbate 20, and methionine and optionally isoleucine. Employing, instead of the animal-derived protein albumin or gelatin, a combination of polysorbate 20 and methionine and optionally isoleucine as  botulinum  toxin stabilizers, the liquid pharmaceutical composition eliminates the risk of contaminating the body with serum-derived pathogens or microorganisms and can be administered safely to the body. Also, the composition is convenient for use as a direct injection for patients. Superior to conventional compositions employing either detergents or amino acids in terms of the storage stability of  botulinum  toxin at 25˜370C as well as at refrigerated temperatures, the liquid pharmaceutical composition of the present invention is very useful for storing  botulinum  toxin under an emergency condition such as an environment without maintaining low temperature. The liquid pharmaceutical composition can be readily prepared because it employs a detergent and an amino acid(s) without a lyophilization process.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a 35 U.S.C. §371 national phase applicationfrom, and claiming priority to, International ApplicationPCT/KR2008/002975, filed May 28, 2008, and published under PCT Article21(2) in English, which claims priority to Korean Application No.10-2007-0069363, filed Jul. 10, 2007, which applications areincorporated by reference herein in their entireties.

TECHNICAL FIELD

The present invention relates, in general, to a more stable liquidpharmaceutical botulinum toxin composition and, more particularly, to aliquid pharmaceutical composition, comprising a botulinum toxin incombination with polysorbate 20 and methionine and optionallyisoleucine.

BACKGROUND ART

Botulinum toxin is a neurotoxin protein produced by the bacteriumClostridium botulinum. This toxin blocks the presynaptic release ofacetylcholine at the neuromuscular junction, causing flaccid (sagging)paralysis of muscles in mammals. The toxin has proven to be effectivefor treating strabismus, idiopathic blepharospasm and hemifacial spasm.In addition, it has recently been found to provide relief for a numberof motor disturbances of involuntary muscles, including spasmodictorticollis, oromandibular dystonia, and spasmodic dyphonia. Further,botulinum toxin received FDA approval for temporary improvement in thefacial appearance of moderate-to-severe frown lines and for thenon-surgical treatment of hyperhidrosis (excessive underarm sweating).

The botulinum toxin proteins serotype A and B are now formulated intodosage forms for use in medical applications such as the treatment oftorticollis, blepharospasm, hyperhidrosis, etc. as well as in cosmeticapplications such as wrinkle reduction. Protein drugs, includingbotulinum toxin proteins, however, suffer from many problems during thepreparation thereof. The problems, most of which are attributed toprotein instability, are particularly pronounced for the protein drugswhich are formulated with very low concentrations of active proteins,such as botulinum toxins.

Adhesing themselves onto solid surfaces, botulinum toxin proteins, whenincased in vessels, are apt to adhere to the inner walls of the vessels,resulting in a loss of the active ingredient. A stabilizing agent isalso required for preventing the proteins from being oxidized ordegraded into fragments.

Albumin is selected in most cases for use as a stabilizer in theformulation of botulinum toxin. In addition to stabilizing the activeprotein ingredients in pharmaceutical compositions, albumin enjoys theadvantage of showing negligible immunogenicity even when injected intothe body. However, serum products such as albumin are not completelyfree from the possibility of being contaminated with pathogens ormicroorganisms and thus acting as mediators of disease, particularlyviral diseases or Creutzfeldt-Jakob disease.

Often, gelatin is employed in place of albumin. Gelatin, however, isrecommended not to be used as a stabilizer for drug formulation becausethis protein, similarly to albumin, is also obtained from animals andmay mediate diseases.

A stabilizer derived from non-animal sources is provided for thepharmaceutical formulation of botulinum toxin by Korean Patent No.10-0799400 in which recombinant serum albumin (rSA), produced in yeast,is applied for pharmaceutical formulation. However, complete avoidancecannot be provided to the possibility that a neoepitope, a new antigenicstructure, may be generated in the course of the production, isolationand recovery of recombinant serum albumin (rSA), eliciting an immuneresponse from the recipient of the drug. Korean Patent No. 10-0665469discloses a pharmaceutical composition comprising a botulinum toxin,polysaccharides (including hydroxyethyl starch) and an amino acid suchas lysine, glycine, histidine or arginine. This pharmaceuticalcomposition is provided in a dosage form prepared by lyophylization andmust unfortunately be stored at low-temperature in a refrigerator orfreezer. The requirement that the lyophilized botulinum toxin be thawedor diluted just before use may cause an error in the protein. Inaddition to being inconvenient for use, lyophilized botulinum toxins aredifficult to develop into a pre-filled syringe administration type.

A composition for stabilization of protein agents in pharmaceuticals issuggested by U.S. Patent Publication No. 2007-0134199. The compositioncomprises a non-ionic detergent, preferably polysorbate, and acombination of either glutamine and glutamic acid or asparagine andaspartic acid. A dilution of botulinum toxin in the liquid compositionwas observed to be stable for 8 months when stored at 4° C. However,because storage at 37° C. decreased the activity of the dilution withinone month, the composition has limited use for the stabilization ofprotein agents, such as botulinum toxin, in pharmaceuticals to be storedat room temperature. This deficit is, in the opinion of the presentinventors, attributed to the presence of the polar amino acids andparticularly the acidic amino acids such as glutamic acid or asparticacid.

DISCLOSURE OF INVENTION Technical Problem

Accordingly, the present invention has been made keeping in mind theabove problems occurring in the prior art, and an object of the presentinvention is to provide a pharmaceutical composition which can maintainthe activity of botulinum toxin even after being stored for a longperiod of time at room temperature as well as at a refrigeratedtemperature and is in a liquid Form more convenient for use than alyophilized form.

It is another object of the present invention to provide a liquidpharmaceutic composition in which the activity of botulinum toxin can bestably maintained under a refrigerated or high temperature conditionwith the use of neither animal-derived protein, such as albumin orgelatin, as a stabilizer for botulinum toxin nor polar or acidic aminoacids such as glutamine, glutamic acid, asparagine or aspartic acid.

Technical Solution

Leading to the present invention, intensive and thorough research,conducted by the present inventors with many detergents and amino acids,into the stable preservation of botulinum toxin under a high-temperaturecondition resulted in the finding that a combination of polysorbate 20and methionine and optionally isoleucine is able to greatly improve thestability of botulinum toxin at room temperature or higher.

In accordance with an aspect of the present invention, there is provideda liquid pharmaceutical composition comprising botulinum toxin,polysorbate 20, and methionine.

In accordance with another aspect of the present invention, there isprovided a liquid pharmaceutical composition comprising botulinum toxin,polysorbate 20, methionine and isoleucine.

In the liquid pharmaceutical composition, the methionine is present inan amount of 0.5 to 100 μmol per 100 units of botulinum toxin, andpreferably ranges in concentration from 0.5 to 100 mM and morepreferably from 25 to 75 mM.

In the liquid pharmaceutical composition according to the presentinvention, polysorbate 20 is present in an amount of 0.01 to 50 mg per100 units of botulinum toxin and preferably ranges in concentration from0.01 to 50 mg/mL and more preferably form 0.1 to 2.5 mg/mL.

In the liquid pharmaceutical composition according to the presentinvention, the botulinum toxin is selected from a group consisting ofbotulinum toxin serotypes A, B, C, D, E, F, and G and may be in anon-complex form or in a complex form with a protein. Preferably, thebotulinum toxin ranges in concentration from 50 to 5,000 units/mL.

The liquid pharmaceutical composition according to the present inventionpreferably ranges in pH from 5.5 to 7.0.

Advantageous Effects

Employing as botulinum toxin stabilizers, instead of the animal-derivedprotein albumin or gelatin, a combination of polysorbate 20 andmethionine, optionally with isoleucine, the liquid pharmaceuticalcomposition according to the present invention eliminates the risk ofcontaminating the body with serum-derived pathogens or microorganismsand can be administered safely.

In addition, the liquid pharmaceutical composition of the presentinvention can be convenient for use as a direct injection for patients.Furthermore, as well as at refrigerated temperatures, in terms of thestorage stability of botulinum toxin at 25˜37° C., the liquidpharmaceutical composition of the present invention is very useful forstoring botulinum toxin under an emergency condition such as anenvironment without maintaining low temperature, thus being superior toconventional liquid pharmaceutical compositions employing eitherdetergents or amino acids.

The liquid pharmaceutical composition of the present invention can bereadily prepared because it employs a detergent and an amino acid(s)without a lyophilization process.

Best Mode For Carrying Out The Invention

A liquid pharmaceutical composition with improvement in botulinumstability is provided in accordance with the present invention.Particularly, the liquid pharmaceutical composition comprises abotulinum toxin, polysorbate 20 and methionine and optionallyisoleucine.

The liquid pharmaceutical composition comprising a botulinum toxin,polysorbate 20 and methionine, or alternatively, a botulinum toxin,polysorbate 20, methionine and isoleucine in accordance with the presentinvention is much improved in botulinum toxin stability.

With the employment of polysorbate 20, methionine and optionallyisoleucine, instead of an animal-derived protein such as albumin orgelatin, as stabilizers for botulinum toxin, the liquid pharmaceuticalcomposition of the present invention excludes the potential risk ofinfecting the recipient with serum-derived pathogens or microorganismsand is thus safe for ingestion into the body. In addition, the use ofthe stabilizers polysorbate 20, methionine and optionally isoleucine incombination guarantees higher stability to botulinum toxin at 25˜27° C.than does the use of them in a separate manner.

The botulinum toxin, a constituent of the liquid pharmaceuticalcomposition according to the present invention, may be one selected fromamong serotypes A, B, C, D, E, F and G. The term botulinum toxin is ageneric term embracing the family of toxins produced by the anaerobicbacterium Clostridium botulinum and, to date, seven immunologicallydistinct neurotoxins serotypes have been identified. These have beengiven the designations A, B, C, D, E, F and G, which differ one from theother in their effects on target animals, and paralysis extent andduration. All serotypes of botulinum toxin are known to act as aneurotoxin by inhibiting the neurotransmitter acetylcholine atneuromuscular junctions.

The botulinum toxin of the liquid pharmaceutical composition accordingto the present invention may be in a non-complex form or in a complexform with another protein. Botulinum toxin serotype A, B, C, D, E, F orG alone, synthesized by Clostridium botulinum, itself has a molecularweight of approximately 150 kDa. When expressed in Clostridiumbotulinum, the botulinum toxin forms various complexes withhemagglutinin proteins and non-hemagglutinin proteins which aid andprotect the activity thereof. Naturally occurring botulinum type Acomplexes have a molecular weight of approximately 900 kDa, 500 kDa or300 kDa. Molecular weights are measured to be approximately 500 kDa forbotulinum toxin type B complexes and type C complexes, approximately 300kDa or 500 kDa for type D complexes, and approximately 300 kDa for typeE and type F complexes.

Although not severely restricted, the concentration of the botulinumtoxin in the liquid pharmaceutical composition of the present inventionpreferably ranges from 50 to 5,000 units/mL depending on the general usethereof.

Methionine is present in an amount from 0.5 to 100 μmol per 100 units ofbotulinum toxin, and preferably ranges in concentration from 0.5 to 100mM and more preferably from 25 to 75 mM in the liquid pharmaceuticalcomposition of the present invention.

One unit (U) of botulinum toxin is defined as the LD₅₀ uponintraperitoneal injection into female Swiss Webster mice weighing 18˜20grams each. The LD₅₀ of botulinum toxin in mice corresponds to about 50picograms.

A methionine content less than 0.5 μmol per 100 units of botulinum toxincannot guarantee the stabilization of the botolinum toxin to a desirablelevel upon long-term storage at room temperature. On the other hand,when methionine is used in an amount exceeding 100 μmol per 100 units ofbotulinum toxin, the excess increment may not promise an additionalstabilization effect in addition to incurring an economic disadvantage.In the liquid pharmaceutical composition of the present invention,methionine properly ranges in concentration from 0.5 to 100 mM when thebotulinum toxin has a concentration of 100 units/mL. Its properconcentration is adjusted to 25˜75 mM in consideration of theconcentration range of polysorbate 20. When the concentration ofmethionine is below 25 mM in the liquid pharmaceutical composition ofthe present invention, its long-term stabilization effect on botulinumtoxin at room temperature does not reach the desirable level, which isobtainable in the proper concentration range of botulinum toxin. On theother hand, a methionine concentration exceeding 75 mM does not provideany additional effect.

In the liquid pharmaceutical composition of the present invention,polysorbate 20 is present in an amount of 0.01 to 50 mg per 100 units ofbotulinum toxin and preferably ranges in concentration from 0.01 to 50mg/mL and more preferably form 0.1 to 2.5 mg/mL.

Polysorbates are a class of emulsifiers used in some pharmaceuticals andin food preparation. They are often used in cosmetics to dissolveessential oils into water-based (oil-in-water) products. There are manykinds of polysorbates that are classified by a number referring to thetotal number of oxyethylene groups, such as polysorbate 20, 40, 60 and80. The liquid pharmaceutical composition of the present inventionemploys polysorbate 20 (commercially available as brand name Tween 20)as a stabilizer for bolulinum toxin.

If the liquid pharmaceutical composition of the present inventioncontains polysorbate 20 in an amount less than 0.01 mg per 100 units ofbotulinum toxin, its long-term stabilization effect on botulinum toxinat room temperature does not reach a desirable level. On the other hand,a polysorbate 20 concentration exceeding 50 mg/mL does not provide anyadditional effect in addition to incurring an economic disadvantage. Ata concentration of 100 units/mL of botulinum toxin in the liquidpharmaceutical composition of the present invention, polysorbate 20 isproperly present in an amount of 0.01˜50 mg/mL and preferably in anamount of 0.1˜2.5 mg/mL when the methionine concentration is taken intoconsideration. When the concentration of polysorbate 20 in the liquidpharmaceutical composition of the present invention is less than 0.1mg/mL, its long-term stabilization effect on botulinum toxin at roomtemperature does not reach a desired level, which is obtainable by thetarget concentration of polysorbate 20. On the other hand, a polysorbate20 concentration exceeding 2.5 mg/mL does not provide any additionaleffect.

In accordance with the present invention, the liquid pharmaceuticalcomposition has a pH of 5.5˜7.0. In the liquid pharmaceuticalcomposition of the present invention adjusted to a pH of 5.5˜7.0,botulinum toxin is stably maintained at room temperature (particularly40° C.) for a long period of time.

MODE FOR THE INVENTION

A better understanding of the present invention may be obtained throughthe following examples which are set forth to illustrate, but are not tobe construed as the limit of the present invention.

1. Experiment Method

(1) Preparation of Liquid Botulinum Toxin Composition

A botulinum toxin was diluted to a final concentration of 100 units/mLin a stabilizing solution.

(2) Stability Assay of Botulinum Toxin

While the prepared liquid botulinum toxin composition was stored at acertain temperature, samples of 1 mL were taken therefrom atpredetermined intervals. 1 mL of the sampled liquid composition was10-fold diluted using 9 mL of an injection solution. The diluted samplewas intraperitoneally injected into five female ICRmice (Institute ofCancer Research, USA) (at a dose of 0.3 mL per mouse, that is, 3units/mouse). While the mice were observed 3 days after theintraperitoneal injection, the death toll and mortality rate wereanalyzed. The liquid botulinum toxin composition was evaluated formaintenance of the activity of botulinum toxin when the mortality ratewas 50% or higher.

2. Selection of Botulinum Toxin Stabilizer

liquid botulinum toxin compositions containing Various candidates ofbotulinum toxin stabilizer were prepared and analyzed for botulinumtoxin stability over time during storage at 25° C. or 37° C. Results ofthe stability experiments at 25° C. and 37° C. are summarized in Tables1 and 2, respectively. In Tables 1 and 2, HSA stands for human serumalbumin and PEG8000 represents polyethylene glycol 8000.

At 25° C., as seen in Table 1, the activity of botulinum toxin wasmaintained for a long period of time in a liquid composition comprisingL-methione(20 mM)+polysorbate20(2 mg/mL)+botulinum toxin(100 units/mL),HSA(5 mg/mL)+polysorbate20(2 mg/mL)+botulinum toxin(100 units/mL),L-Isoleucine(50 mM)+polysorbate 20(2 mg/mL)+botulinum toxin(100units/mL), a Hydroxyethyl starch(10 mg/mL)+polysorbate 20(2mg/mL)+botulinum toxin(100 units/mL). At 37° C., the liquid compositioncomprising L-methione(20 mM)+polysorbate 20(2 mg/mL)+botulinum toxin(100units/mL) or FISA(5 mg/mL)+polysorbate 20(2 mg/mL)+botulinum toxin(100units/mL) was found to maintain the activity of botulinum toxin for along period of time as seen in Table 2.

From the results, it is recognized that a combination of methionine andpolysorbate 20 acts as a stabilizer substitutable for a combination ofHSA and polysorbate 20 or a combination of hydroxyethyl starch andpolysorbate 20. Also, a combination of isoleucine and polysorbate 20emerged as a stabilizer candidate substitutable for conventionalstabilizers.

TABLE 1 Botulinum Toxin Composition Ex. Stabilizer PolySor B. toxinMortality rate(%) Nos. (Conc.) 20(mg/mL) (Units/mL) Day 0 Day 6 Day 14Day 23 Day 35 1 L-Met (20 mM) 2.0 100 100 100 100 100 100 C. 1 — 2.0 100100 0 — — — C. 2 HSA (5 mg/mL) — 100 100 100 0 — — C. 3 HSA (5 mg/mL)2.0 100 100 100 100 100 100 C. 4 L-Arg(50 mM) 2.0 100 100 0 — — — C. 5L-Ile(50 mM) 2.0 100 100 80 100  60  80 C. 6 L-Glu(50 mM) 2.0 100 100100 0 — — C. 7 His(10 mM) 2.0 100 100 0 — — — C. 8 Glu(8 mg/ml) 2.0 100100 100 60  0 — C. 9 Gly(2 mg/ml) 2.0 100 100 0 — — — C. 10 Na Glm(10mM) 2.0 100 100 100 0 — — C. 11 Hydroxyethyl 2.0 100 100 100 80  80  60Starch(10 mg/ml) C. 12 Mannitol 2.0 100 100 0 — — — (50 mg/ml) C. 13Sorbitol 2.0 100 100 60 0 — — (50 mg/ml) C. 14 C8-Glucose 2.0 100 100 0— — — (50 mg/ml) C. 15 C8-Trehalose 2.0 100 100 0 — — — (50 mg/ml) C. 16Sucrose 2.0 100 100 0 — — — (50 mg/ml) C. 17 Lactose 2.0 100 100 0 — — —(50 mg/ml) C. 18 PEG8000 2.0 100 100 0 — — — (100 mg/ml) C. 19Hyaluronic 2.0 100 100 0 — — — acid (10 mg/ml)

TABLE 2 Botulinum Toxin Composition Ex. Stabilizer PolySor B. toxinMortality rate(%) Nos. (Conc.) 20(mg/mL) (Units/mL) Day 0 Day 6 Day 14Day 23 Day 35 1 L-Met (20 mM) 2.0 100 100 100 100  100 100 C. 1 — 2.0100 100 0 — — — C. 2 HSA (5 mg/mL) — 100 100 100 0 — — C. 3 HSA (5mg/mL) 2.0 100 100 100 80  100 100 C. 4 L-Arg(50 mM) 2.0 100 100 0 — — —C. 5 L-Ile(50 mM) 2.0 100 100 0 — — — C. 6 L-Glu(50 mM) 2.0 100 100 0 —— — C. 7 His(10 mM) 2.0 100 100 0 — — — C. 8 Glu(8 mg/ml) 2.0 100 100 0— — — C. 9 Gly(2 mg/ml) 2.0 100 100 0 — — — C. 10 Na Glm(10 mM) 2.0 100100 0 — — — C. 11 Hydroxyethyl 2.0 100 100 100 0 — — Starch(10 mg/ml) C.12 Mannitol 2.0 100 100 0 — — — (50 mg/ml) C. 13 Sorbitol 2.0 100 100 0— — — (50 mg/ml) C. 14 C8-Glucose 2.0 100 100 0 — — — (50 mg/ml) C. 15C8-Trehalose 2.0 100 100 0 — — — (5 mg/ml) C. 16 Sucrose 2.0 100 100 0 —— — (50 mg/ml) C. 17 Lactose 2.0 100 100 0 — — — (50 mg/ml) C. 18PEG8000 2.0 100 100 0 — — — (100 mg/ml) C. 19 Hyaluronic 2.0 100 100 0 —— — acid (10 mg/ml)

3. Stability of Botulinum Toxin at Various Concentrations of Methionine

Liquid botulinum toxin compositions comprising various concentrations ofmethionine in combination with polysorbate 20 as botulinum toxinstabilizers were assayed for ability to maintain the activity ofbotulinum toxin under a 37° C. storage condition. The experimentalresults of the stability of botulinum toxin according to a change inmethionine concentration are summarized in Table 3, below.

TABLE 3 Botulinum Toxin Composition Mortality rate(%) Ex. Met PolySor B.toxin Day Nos. (mM) 20(mg/mL) (Units/mL) 4 10 18 24 29 44 56 70 2 0.52.0 100 100 100 100 100 100 100 60 0 3 1.0 2.0 100 100 100 100 100 100100 100 100 4 20 2.0 100 100 100 100 100 100 100 100 0 5 50 2.0 100 100100 100 100 100 100 100 100 6 100 2.0 100 100 100 100 100 100 100 100100 C. 20 — 2.0 100 0 — — — — — — —

As seen in Table 3, the activity of botulinum toxin was stablymaintained for 56 days at a methionine concentration of 0.5˜20 mM andfor 70 days at a methionine concentration of 50˜100 mM. The compositionemploying both methionine and polysorbate 20 greatly improved thestability of botulinum toxin as compared with the composition employingpolysorbate 20 alone.

4. Stability of Botulinum Toxin at Various Concentrations of Polysorbate20

Liquid botulinum toxin compositions comprising methionine in combinationwith various concentrations of polysorbate 20 as botulinum toxinstabilizers were assayed for ability to maintain the activity ofbotulinum toxin under a 37° C. storage condition. The experimentalresults of the stability of botulinum toxin according to a change inpolysorbate concentration are summarized in Table 4, below.

TABLE 4 Botulinum Toxin Composition Mortality rate(%) Ex. Met PolySor B.toxin Day Nos. (mM) 20(mg/mL) (Units/mL) 30 60 90 120 167 202  7 20 0.01100 100 100 100 100 80 0  8 20 0.5 100 100 80 100 100 80 100  9 20 2.0100 100 100 80 100 80 100 10 20 10 100 100 100 80 100 40 0 11 20 50 100100 0 — — — — C. 21 1 — 100 0 — — — — — C. 22 5 — 100 0 — — — — — C. 2310 — 100 0 — — — — — C. 24 20 — 100 0 — — — — —

As seen in Table 4, the activity of botulinum toxin was stablymaintained for 202 days at a polysorbate 20 concentrations of 0.5˜2.0mg/mL, for 167 days at a polysorbate 20 concentration of 0.01 mg/mL andfor 120 days at a polysorbate 20 concentration of 10 mg/mL. Thecomposition employing both methionine and polysorbate 20 was greatlyimproved in the stability of botulinum toxin as compared with thecomposition employing methionine alone.

5. Stability of Botulinum Toxin at Various Concentrations of Methionineand Polysorbate 20

Liquid botulinum toxin compositions comprising various concentrations ofa combination of methionine and polysorbate 20 as botulinum toxinstabilizers were assayed for their ability to maintain the activity ofbotulinum toxin under a 37° C. storage condition. The experimentalresults (mortality rate, %)of the stability of botulinum toxin afterstorage for 30 and 60 days at various concentrations of methionine andpolysorbate 20 (Example 12) are summarized in Tables 5 and 6,respectively. In the liquid botulinum toxin compositions used in theexperiments, botulinum toxin had a concentration of 100 units/mL.

Statistical analysis of the data of Tables 5 and 6 suggests that acombination of 25˜75 mM of methionine and 0.1˜2.5 mg/mL of polysorbate20 stabilize botulinum toxin at highest efficiency.

TABLE 5 Conc. Of Met (mM) Polysorbate 20/Met 1 5 10 25 50 75 100Polysorbate 20 0.1 100 100 80 100 100 100 100 (mg/mL) 0.5 100 100 80 100100 100 100 2.5 100 100 100 100 100 100 100 10 100 100 100 100 100 100100 25 100 100 100 100 100 100 100 50 80 100 100 80 80 100 100

TABLE 6 Conc. Of Met (mM) Polysorbate 20/Met 1 5 10 25 50 75 100Polysorbate 20 0.1 100 100 80 100 100 80 100 (mg/mL) 0.5 100 100 80 100100 100 100 2.5 100 100 100 80 100 100 100 10 0 40 0 100 100 100 40 25 080 80 100 — 100 60 50 0 0 0 0 80 — 0

6. Stability of Botulinum Toxin in Liquid Botulinum Toxin Compositionswith Various pHs

Various liquid botulinum toxin compositions (Example 13)with a pH of5.5˜7.0, in which methionine and polysorbate 20 were combined in theirrespective concentration ranges optimal for the stabilization ofbotulinum toxin, were prepared and assayed for ability to maintain theactivity of botulinum toxin under a 40° C. storage condition. The pH ofthe liquid botulinum toxin compositions was adjusted with HCl or NaOH.Each of the compositions had a botulinum toxin concentration of 100units/mL. The results of the stability of botulinum toxin according tothe pH of the liquid composition are summarized in Table 7, below.

As seen in Table 7, the activity of botulinum toxin was stablymaintained for 90 days in liquid botulinum toxin compositions containingof 25˜75 mM of methionine in combination with 0.25 ˜50.75 mg/mL ofpolysorbate 20 with the pH thereof ranging from 5.5 to 7.0 under a 40°C. storage condition.

TABLE 7 Botulinum Toxin Composition Mortality rate(%) Met PolySorbate20Day (mM) (mg/mL) pH 7 15 30 60 90 25 0.25 5.5 100 100 100 100 100 6.0100 100 100 100 100 6.5 100 100 100 100 100 7.0 100 100 100 100 100 0.55.5 100 100 100 100 100 6.0 100 100 100 100 100 6.5 100 100 100 100 1007.0 100 100 100 100 100 0.75 5.5 100 100 100 100 100 6.0 100 100 100 100100 6.5 100 100 100 100 100 7.0 100 100 100 100 100 50 0.25 5.5 100 100100 100 100 6.0 100 100 100 100 100 6.5 100 100 100 100 100 7.0 100 100100 100 100 0.5 5.5 100 100 100 100 100 6.0 100 100 100 100 100 6.5 100100 100 100 100 7.0 100 100 100 100 100 0.75 5.5 100 100 100 100 100 6.0100 100 100 100 100 6.5 100 100 100 100 100 7.0 100 100 100 100 100 750.25 5.5 100 100 100 100 100 6.0 100 100 100 100 100 6.5 100 100 100 100100 7.0 100 100 100 100 100 0.5 5.5 100 100 100 100 100 6.0 100 100 100100 100 6.5 100 100 100 100 100 7.0 100 100 100 100 100 0.75 5.5 100 100100 100 100 6.0 100 100 100 100 100 6.5 100 100 100 100 100 7.0 100 100100 100 100

7. Stability of Botulinum Toxin in Liquid Pharmaceutical CompositionContaining a Combination of Methionine, Isoleucine and Polysorbate

As described above, a combination of isoleucine and polysorbate 20 aswell as a combination of methione and polysorbate 20 was identified as acandidate for stabilizing botulinum toxin. On the basis of this result,a combination of methionine, isoleucine and polysorbate 20 was assayedfor ability to stabilize botulinum toxin under a 37° C. storagecondition. In the liquid composition, botulinum toxin had aconcentration of 100 units/mL.

TABLE 8 Botulinum Toxin Composition Mortality rate(%) Ex. Met IlePolysorbate20 Day Nos. (mM) (mg/mL) (mg/mL) 30 60 90 120 167 202 14 2010 0.5 100 100 80 100 100 100 15 20 10 2.0 100 80 80 100 100 0 16 20 1010 100 100 100 100 100 100 C. 25 — 10 — 80 0 — — — — C. 26 20 10 — 0 — —— — — C. 27 — 10 0.5 0 — — — — — C. 28 — 1 2.0 0 — — — — — C. 29 — 102.0 0 — — — — — C. 30 — 30 2.0 0 — — — — — C. 31 — 50 2.0 0 — — — — — C.32 — 100 2.0 0 — — — — — C. 33 — 10 10 0 — — — — —

As seen in Table 8, the activity of botulinum toxin was maintained for along period of time (approximately 200 days) under a 37° C. storagecondition by a combination of methionine, isoleucine and polysorbate 20,but was found to almost disappear before the lapse of 30 days in theliquid compositions containing isoleucine alone or in combination withmethionine or polysorbate 20.

Industrial Applicability

As described hitherto, the liquid pharmaceutical botulinum toxincomposition according to the present invention shows greatly improvedbotulinum toxin stability. It is useful in the treatment of dystonia,stiff muscle spasm, neurological disorders (migraine, lumbago, cervicalspinal disorder, etc.) as well as in the cosmetic application forhyperhidrosis treatment and wrinkle reduction. In addition, the liquidpharmaceutical botulinum toxin composition of the present invention maybe directly used as an injection and guarantees the activity ofbotulinum toxin for a long period of time even at 25˜37° C. as well asat a refrigerated temperature, which is very advantageous fortransportation and sale.

The invention claimed is:
 1. A liquid pharmaceutical compositioncomprising botulinum toxin, polysorbate 20, and methionine, wherein themethionine ranges in concentration from 0.5 to 100 mM, the polysorbate20 is present in an amount of 0.01 to 50 mg per 100 units of botulinumtoxin, and wherein the composition is free of gelatin or human serumalbumin (HSA).
 2. The liquid pharmaceutical composition according toclaim 1, wherein the methionine ranges in concentration from 25 to 75mM.
 3. The liquid pharmaceutical composition according to claim 1,wherein the polysorbate 20 ranges in concentration from 0.01 to 50mg/mL.
 4. The liquid pharmaceutical composition according to claim 3,wherein the polysorbate 20 ranges in concentration from 0.1 to 2.5mg/mL.
 5. The liquid pharmaceutical composition according to claim 1,wherein the botulinum toxin is selected from a group consisting ofbotulinum toxin serotypes A, B, C, D, E, F, and G.
 6. The liquidpharmaceutical composition according to claim 1, wherein the botulinumtoxin is in a non-complex form or in a complex form with hemagglutininproteins (HA) or non-toxic non-hemagglutinin proteins (NTNH).
 7. Theliquid pharmaceutical composition according to claim 1, wherein theliquid pharmaceutical composition ranges in pH from 5.5 to 7.0.
 8. Theliquid pharmaceutical composition according to claim 1, furthercomprising isoleucine.
 9. The liquid pharmaceutical compositionaccording to claim 1, wherein the botulinum toxin ranges inconcentration from 50 to 5,000 units/mL.